The objective of this research are to further purify and characterize exonuclease VII of E. coli and to examine its role in DNA replication, recombination and repair. The work will exploit mutants which I have isolated having reduced levels of exonuclease VII activity to study 1) the joining of Okazaki fragments; 2) excision of thymine dimers and repair synthesis and 3) whether or not a single protein catalyzes the hydrolysis of single stranded DNA from both 5' and 3' termini. The previous work that I have done on this enzyme has resulted in a 1700 fold purification. I will now purify it to hemogeneity and study its subunit structure. The homogeneous enzyme will be used in in vitro studies employing model compounds that may be structurally similar to intermediates in DNA replication and recombination to test mechanisms by which exonuclease VII could function in these processes according to current models. Studies will be also carried out related to the practical application of exonuclease VII as a general enzymatic reagent for certain types of modifications of polynucleotide structures. In addition, we will attempt to develop a new assay for exonuclease VII and other nucleases whose mechanisms of action are similar to that of exonuclease VII (specifically those that release large, acid-insoluble initial products).